The goal of this project is to characterize the Treg in SLE and RA, both quantitatively and in their regulatory effector function. The ability to induce or expand Treg cells in vivo and in vitro could have important implications not only in the field of autoimmunity, but also in transplantation tolerance. An important advantage is that because Treg cells can exert bystander suppression in a non antigen-specific manner, they need not necessarily recognize the target antigen(s) that are the subject of immune attack. Induction of Treg cells that react to any local tissue?expressed molecule may be sufficient to inhibit immune. With these goals in mind, the initial emphasis was to develop the means to assess the human immune system, with the understanding that the source of potential sampling was limited and the amounts of material was often limited. Experiments carried out have examined the phenotype and function of CD4+CD25+ regulatory T cells in normals and patients with autoimmune diseases, including SLE and rheumatoid arthritis. By using the bright expression of CD25, a population enriched in Tregs were obtained that suppressed the proliferation of CD4+ T cells after anti-CD3 stimulation in vitro and also the production of IFNg. Withe recent availavility of FoxP3 mAb we have further confimed that the selected CD4+CD25bright T cells are true Tregs as they expressed high levels og FoxP3 in healthy controls. Suppression could not be accounted for by the production of cytokines (IL-4, IL-10 or TGFb), but appeared to be contact dependent, as previously reported in humans and mice. This population of Tregs constitutively expressed high levels of FoxP3 mRNA, and protein whereas the CD4+ effector cells did not express FoxP3. It was notable that GITR was only modestly expressed constitutively by Tregs and appeared to play no role in modulating the function of Tregs or the responsiveness of effector cells to suppression by Tregs. Analysis of the detailed phenotype revealed that human Tregs demonstrated that their expressed increased levels of TNFRII, but no TNFRI. Moreover, addition of TNF to cultures of Tregs and effector cells completely abolished inhibition of responsiveness. Similar results were noted with an agonistic monoclonal antibody to TNFRII. To document that TNF influenced the behavior of Tregs, directly, CD4+CD25+ cells were isolated and cultured with IL-2 alone or IL-2 in combination with TNF. Afterward the cells were washed, cultured with effector cells and stimulated with ant-CD3. Preincubation with TNF completely blocked the capacity of Tregs to suppress proliferation of IFNgamma production by CD4+effector cells. Similar results were noted when Tregs were pre-incubated with an agonistic monoclonal antibody to TNFRII. Moreover, preincubation with either TNF or the antibody to TNFRII significantly downregulated expression of FoxP3 mRNA. In contrast, preincubation with TNF had no effect on the capacity of CD4+ effector cells to be suppressed by Tregs. These data strongly indicate that TNF can down-regulate the function of Tregs by a mechanism that involves signals triggered by engagement of TNFRII. To determine whether Tregs are defective in patients with inflammatory disease and elevated levels of TNF, patients with both rheumatoid arthritis and systemic lupus erythematosus were examined. Both patient groups manifested normal proportions of CD25+CD4+ T cells but defective suppressive activity. Moreover, constitutive expression of FOXP3 mRNA was significantly diminished. In each circumstance, the defective suppression was reversed by in vitro stimulation with anti-CD3 and IL-2. This was accompanied by an up-regulation of FoxP3 mRNA. Notably, TNF and the antibody to TNFRII blocked the normalization of suppressive function. Importantly, a number of patients with rheumatoid arthritis were analyzed before and after therapy with a therapeutic anti-TNF specific monoclonal antibody (infliximab). The suppression of Treg function and decreased FoxP3 expression were both reversed after therapy with infliximab, indicating that TNF inhibited Treg function by downmoduating FoxP3 expression in vivo. [unreadable] These experiments have provided the basis for further analysis of the regulation of Treg function and also the basis to consider therapeutic interventions aimed at augmenting Treg function in patients with autoimmune disease. Initially, we will focus on the mechanism by with TNF regulates FoxP3 expression in Tregs. In humans, the agonistic antibody to TNFRII gives us the opportunity to study the impact of TNF signaling on FoxP3 expression at the mRNA level as well as the protein level using recently released monoclonal antibodies to FoxP3 and western blotting and eventually flow cytometry when monoclonal antibodies appropriate for this purpose become available.To pursue this further, we have established a collaboration with Ethan Shevach to assess the impact of TNF on well established models of murine Treg function using various genetically altered mice including those lacking TNF, TNFRI and TNFRII. Our major focus is to develop these initial finding into a clinical trial. [unreadable] Goals for next year:[unreadable] 1. Isolate Tregs, from lupus patients, activate them in vitro with beads coated with anti-CD3 and anti-CD28 obtained by an MTA with XCYTE along with IL2 and expand them under cGMP conditions with colleagues from the Transfusion Medicine Service of the Clinical Center. Initially these will be transfused back into autologous patients in a phase I study. The primary goal of the study is to monitor the safety of the procedure, but we will also monitor the persistence of the activated T regs as well as any clinical benefit. If safe, we plan to pursue this approach in subsequent trials to determine the potential efficacy of transfusion of in vitro activated and expanded Tregs. The second approach involves attempting to modulate Treg function in vivo by blocking TNF activity. We have begun to monitor Treg function in patients with rheumatoid arthritis treated with infliximab to determine whether the gain of Treg function correlates with the clinical improvement. In a second approach we are considering a similar protocol in patients with systemic lupus erythematosus. Even though preliminary data suggest that this can be done safely in patients receiving concomitant immunosuppressive agents, there remains the danger that blocking TNF will exacerbate disease anti -TNF therapy is associated with an increase in autoantibodies to DNA in approximately 10% of treated patients. For other purposes, we have been comtemplating a study of sequential treatment with rituximab to deplete B cell followed by placebo or anti-TNF to modify ongoing inflammation. A course of anti-TNF should be safer in patients following B cell depletion.This trial will give us an ideal opportunity to determine whether anti-TNF therapy is associated with accentuated Treg function in lupus patients and whether this is associated with sustained improvement in disease activity.